Lactoferrin

ABSTRACT

A pure lactoferrin polypeptide containing no more than two metal ions per molecule, or a mixture of the polypeptide and a fragment thereof. The polypeptide or the mixture stimulates skeletal growth and inhibits bone resorption. Also disclosed is a method of treating a bone-related disorder with the polypeptide or the mixture.

RELATED APPLICATION

[0001] This application claims priority to New Zealand ApplicationSerial No. 518121, filed Apr. 3, 2002, the content of which isincorporated herein by reference.

BACKGROUND

[0002] Lactoferrin is an 80 kD iron-binding glycoprotein present in mostexocrine fluids, including tears, bile, bronchial mucus,gastrointestinal fluids, cervico-vaginal mucus, seminal fluid, and milk.It is a major constituent of the secondary specific granules ofcirculating poly-morphonuclear neutrophils. The richest source oflactoferrin is mammalian milk and colostrum.

[0003] Lactoferrin circulates at a concentration of 2-7 μg/ml. It hasmultiple postulated biological roles, including regulation of ironmetabolism, immune function, and embryonic development. Lactoferrin hasanti-microbial activity against a range of pathogens including Grampositive and Gram negative bacteria, yeasts, and fungi. Theanti-microbial effect of lactoferrin is based on its capability ofbinding iron, which is essential for the growth of the pathogens.Lactoferrin also inhibits the replication of several viruses andincreases the susceptibility of some bacteria to antibiotics andlysozyme by binding to lipid A component of lipopolysaccharides onbacterial membranes.

SUMMARY

[0004] This invention relates to a lactoferrin polypeptide that iscapable of stimulating skeletal growth and inhibiting bone resorption.

[0005] Specifically, this invention features a pure lactoferrinpolypeptide containing no more than two (i.e., 0, 1, or, preferably, 2)metal ions per molecule. A “pure” polypeptide is a polypeptide free fromother biological macromolecules and at least 65% (e.g., at least 70, 75,80, 85, 90, 95, or 99%) pure by dry weight. The purity of a polypeptidecan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis. The lactoferrin polypeptide can be a naturally occurringpolypeptide, a recombinant polypeptide, or a synthetic polypeptide.Variants of a wild-type lactoferrin polypeptide (e.g., a fragment of thewild-type lactoferrin polypeptide containing at least 2 (e.g., 4, 6, 8,10, 20, 50, 100, 200, 300, 400, 500, 600, 700) amino acids, or arecombinant protein containing a lactoferrin polypeptide sequence) thatmaintain the biological activity of a wild-type lactoferrin polypeptideare within the scope of the invention. A lactoferrin polypeptide of theinvention can be of a mammalian origin, e.g., from human or bovine milk.The metal ion bound to the polypeptide can be an iron ion (as in anaturally occurring lactoferrin polypeptide), a copper ion, a chromiumion, a cobalt ion, a manganese ion, a zinc ion, or a magnesium ion.

[0006] A lactoferrin polypeptide of the invention can be used tostimulate skeletal growth (e.g., by promoting proliferation ofosteoblasts and chondrocytes) and inhibit bone resorption (e.g., byinhibiting osteoclast development). A preparation of a lactoferrinpolypeptide of the invention (e.g., lactoferrin isolated from bovinemilk) can contain polypeptides of a single species, e.g., every moleculebinding two iron ions. It can also contain polypeptides of differentspecies, e.g., some molecules binding no ion and others each binding oneor two ions; some molecules each binding an iron ion and others eachbinding a copper ion; some molecules each being a biological activelactoferrin polypeptide (full-length or shorter than full-length) thatcontains 0, 1, or 2 metal ions and others each being a fragment (same ordifferent) of the polypeptide; or all molecules each being a fragment(same or different) of a full-length lactoferrin polypeptide thatcontains 0, 1, or 2 metal ions. For example, a mixture of full-lengthlactoferrin polypeptides and various fragments of full-lengthlactoferrin polypeptides can be prepared from a hydrolysate, e.g., apartial digest such as a proteinase digest, of full-length lactoferrinpolypeptides. Otherwise, it can be obtained by mixing full-lengthlactoferrin polypeptides with various fragments of full-lengthlactoferrin polypeptides (e.g., synthetic fragments). A mixture ofvarious fragments of full-length lactoferrin polypeptides, on the otherhand, can be prepared, for example, by complete digestion (i.e., nofull-length polypeptides remain after digestion) of full-lengthlactoferrin polypeptides, or by mixing different fragments offull-length lactoferrin polypeptides.

[0007] The invention further features a nutraceutical composition, whichcan be milk, juice, a soft drink, a snack bar, or a dietary supplement.The nutraceutical composition contains a lactoferrin polypeptide of theinvention or a mixture of the polypeptide and fragments of thepolypeptide in an amount higher than the naturally occurring amount.Lactoferrin has been found to stimulate osteoblast and chondrocyteproliferation and inhibit osteoclast development. Thus, a nutraceuticalcomposition of this invention is useful for preventing and treating bonedisorders such as osteoporosis and rheumatoid or osteo-arthritis. Thenutraceutical composition can further include an adequate amount ofanother bone-enhancing agent, such as calcium, zinc, magnesium, vitaminC, vitamin D, vitamin E, vitamin K2, or a mixture thereof.

[0008] In addition, this invention features a pharmaceutical compositionthat contains a lactoferrin polypeptide of the invention or a mixture ofthe polypeptide and fragments of the polypeptide and a pharmaceuticallyacceptable carrier. Optionally, the pharmaceutical composition alsoincludes another bone-enhancing agent. The invention also encompassesthe use of a lactoferrin polypeptide or a mixture of the polypeptide andfragments of the polypeptide described above for the manufacture of amedicament for preventing and treating bone diseases.

[0009] This invention provides a method of preventing and treatingbone-related disorders (e.g., by stimulating skeletal growth andinhibiting bone resorption). The method includes administering to asubject in need thereof an effective amount of a lactoferrin polypeptideof the invention or a mixture of the polypeptide and fragments of thepolypeptide. The method can further include concurrently administeringto the subject an effective amount of another bone-enhancing agent.

[0010] The details of one or more embodiments of the invention are setforth in the accompanying description below. Other features, objects,and advantages of the invention will be apparent from the detaileddescription, and from the claims.

DETAILED DESCRIPTION

[0011] This invention is based on the unexpected discovery thatlactoferrin stimulates osteoblast and chondrocyte proliferation andinhibits osteoclast development. Thus, it is useful for preventing andtreating bone disorders.

[0012] A lactoferrin polypeptide of the invention is a pure polypeptidecontaining no more than two metal ions per molecule. Practically, themeasurement of the ion/lactoferrin ratio for a preparation oflactoferrin can be in the range of 0-2.5. It can be isolated from anatural source (e.g., mammalian milk), or produced using geneticengineering or chemical synthesis techniques well-known in the art. Thefollowing is an exemplary procedure for isolating lactoferrin frombovine milk:

[0013] Fresh skim milk (7 L, pH 6.5) is passed through a 300 ml columnof S Sepharose Fast Flow equilibrated in milli Q water, at a flow rateof 5 ml/min and at 4° C. Unbound protein is washed through with 2.5 bedvolumes of water and bound protein eluted stepwise with approximately2.5 bed volumes each of 0.1 M, 0.35 M, and 1.0 M sodium chloride.Lactoferrin eluting as a discreet pink band in 1 M sodium chloride iscollected as a single fraction and dialysed against milli Q waterfollowed by freeze-drying. The freeze-dried powder is dissolved in 25 mMsodium phosphate buffer, pH 6.5 and subjected to rechromatography on SSepharose Fast Flow with a sodium chloride gradient to 1 M in the abovebuffer and at a flow rate of 3 ml/min. Fractions containing lactoferrinof sufficient purity as determined by gel electrophoresis and reversedphase HPLC are combined, dialyzed and freeze-dried. Final purificationof lactoferrin is accomplished by gel filtration on Sephacryl 300 in 80mM dipotassium phosphate, pH 8.6, containing 0.15 M potassium chloride.Selected fractions are combined, dialyzed against milli Q water, andfreeze-dried. The purity of this preparation is greater than 95% asindicated by HPLC analysis and by the spectral ratio values (280 nm/465nm) of ˜19 or less for the iron-saturated form of lactoferrin.

[0014] Iron saturation is achieved by addition of a 2:1 molar excess of5 mM ferric nitrilotriacetate (Foley and Bates (1987) AnalyticalBiochemistry 162, 296-300) to a 1% solution of the purified lactoferrinin 50 mM Tris, pH 7.8 containing 10 mM sodium bicarbonate. Excess ferricnitrilotriacetate is removed by dialysis against 100 volumes of milli Qwater (twice renewed) for a total of 20 hours at 4° C. The iron-loaded(holo-) lactoferrin is then freeze-dried.

[0015] Iron-depleted (apo-) lactoferrin is prepared by dialysis of a 1%solution of the highly purified lactoferrin sample in water against 30volumes of 0.1 M citric acid, pH 2.3, containing 500 mg/L disodium EDTA,for 30 h at 4° C. (Massons and Heremans (1966) Protides of theBiological fluids 14, 115-124). Citrate and EDTA are then removed bydialysis against 30 volumes of milli Q water (once renewed) and theresulting colourless solution freeze-dried.

[0016] A lactoferrin polypeptide of the invention can contain an ironion (as in a naturally occurring lasctoferrin polypeptide) or a non-ironmetal ion (e.g., a copper ion, a chromium ion, a cobalt ion, a manganeseion, a zinc ion, or a magnesium ion). For instance, lactoferrin isolatedfrom bovine milk can be depleted of iron and then loaded with anothertype of metal ion. For example, copper loading can be achieved accordingto the same method for iron loading described above. For loadinglactoferrin with other metal ions, the method of Ainscough, et al.((1979) Inorganica Chimica Acta 33, 149-153) can be used.

[0017] In a preparation of a lactoferrin polypeptide of the invention,the polypeptides can be of a single species, or of different species.For instance, the polypeptides can each contain a different number ofmetal ions or a different species of metal ions; or the lengths of thepolypeptides can vary, e.g., some are full-length polypeptides and someare fragments, and the fragments can each represent a particular portionof a full-length polypeptide. Such a preparation can be obtained from anatural source or by mixing different lactoferrin polypeptide species.For example, a mixture of lactoferrin polypeptides of different lengthscan be prepared by proteinase digestion (complete or partial) offull-length lactoferrin polypeptides. The degree of digestion can becontrolled according to methods well known in the art, e.g., bymanipulating the amount of proteinase or the time of incubation. Acomplete digestion produces a mixture of various fragments offull-length lactoferrin polypeptides; a partial digestion produces amixture of full-length lactoferrin polypeptides and various fragments.

[0018] A lactoferrin polypeptide or a mixture of the polypeptide andfragments of the polypeptide described above is used to prepare anutraccutical composition of this invention for preventing and treatingbone-related disorders. Examples of such disorders include, but are notlimited to, osteoporosis, rheumatoid or osteo-arthritis, hepaticosteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renalosteodystrophy, osteosclerosis, osteopenia, fibrogenesis-imperfectaossium, secondary hyperparathyrodism, hypoparathyroidism,hyperparathyroidism, chronic renal disease, sarcoidosis,glucocorticoid-induced osteoporosis, idiopathic hypercalcemia, Paget'sdisease, and osteogenesis imperfecta. The nutraceutical composition canbe a dietary supplement (e.g., a capsule, a mini-bag, or a tablet) or afood product (e.g., milk, juice, a soft drink, a herbal tea-bag, orconfectionary). The composition can also include other nutrients, suchas a protein, a carbohydrate, vitamins, minerals, or amino acids. Thecomposition can be in a form suitable for oral use, such as a tablet, ahard or soft capsule, an aqueous or oil suspension, or a syrup; or in aform suitable for parenteral use, such as an aqueous propylene glycolsolution, or a buffered aqueous solution. The amount of the activeingredient in the nutraceutical composition depends to a large extent ona subject's specific need. The amount also varies, as recognized bythose skilled in the art, dependent on administration route, andpossible co-usage of other bone-enhancing agents.

[0019] Also within the scope of this invention is a pharmaceuticalcomposition that contains an effective amount of a lactoferrinpolypeptide or a mixture of the polypeptide and fragments of thepolypeptide described above, and a pharmaceutically acceptable carrier.The pharmaceutical composition can be used to prevent and treatbone-related disorders described above. The pharmaceutical compositioncan further include an effective amount of another bone-enhancing agent.The pharmaceutically acceptable carrier includes a solvent, a dispersionmedium, a coating, an antibacterial and antifungal agent, and anisotonic and absorption delaying agent. An “effective amount” is theamount required to confer therapeutic effect. The interrelationship ofdosages for animals and humans (based on milligrams per meter squared ofbody surface) is described by Freireich, et al. (1966) Cancer Chemother.Rep. 50: 219. Body surface area can be approximately determined fromheight and weight of the subject. See, e.g., Scientific Tables, GeigyPharmaceuticals, Ardley, N. Y., 1970, 537. Effective doses also vary, asrecognized by those skilled in the art, dependent on route ofadministration, excipient usage, and the like.

[0020] A lactoferrin polypeptide of the invention or a mixture of thepolypeptide and fragments of the polypeptide can be formulated intodosage forms for different administration routes utilizing conventionalmethods. For example, it can be formulated in a capsule, a gel seal, ora tablet for oral administration. Capsules can contain any standardpharmaceutically acceptable materials such as gelatin or cellulose.Tablets can be formulated in accordance with conventional procedures bycompressing mixtures of the lactoferrin polypeptide or a mixture of thepolypeptide and fragments of the polypeptide with a solid carrier and alubricant. Examples of solid carriers include starch and sugarbentonite. The lactoferrin polypeptide or a mixture of the polypeptideand fragments of the polypeptide can also be administered in a form of ahard shell tablet or a capsule containing a binder, e.g., lactose ormannitol, a conventional filler, and a tableting agent. Thepharmaceutical composition can be administered via the parenteral route.Examples of parenteral dosage forms include aqueous solutions, isotonicsaline or 5% glucose of the active agent, or other well-knownpharmaceutically acceptable excipient. Cyclodextrins, or othersolubilizing agents well-known to those familiar with the art, can beutilized as pharmaceutical excipients for delivery of the therapeuticagent.

[0021] The efficacy of a composition of this invention can be evaluatedboth in vitro and in vivo. See, e.g., the examples below. Briefly, thecomposition can be tested for its ability to promote osteoblast andchondrocyte proliferation in vitro. For in vivo studies, the compositioncan be injected into an animal (e.g., a mouse) and its effects on bonetissues are then accessed. Based on the results, an appropriate dosagerange and administration route can be determined.

[0022] The specific examples below are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. All publications recited hereinare hereby incorporated by reference in their entirety.

[0023] Lactoferrin Promotes Proliferation of Primary Rat Osteoblasts

[0024] Osteoblasts were isolated by collagenase digestion from 20-dayfetal rat calvariae, as previously described by Lowe and co-workers(Lowe, et al. (1991) Journal of Bone and Mineral Research 6, 1277-1283).Calvariae were dissected aseptically, and the frontal and parietal boneswere stripped of their periosteum. Only the central portions of thebones, free from suture tissue, were collected. The calvariae weretreated twice with phosphate buffered saline (PBS) containing 3 mM EDTA(pH 7.4) for 15 minutes at 37° C. in a shaking water bath. After washingonce in PBS, the calvariae were treated twice with 3 ml of 1 mg/mlcollagenase for 7 minutes at 37° C. After discarding the supernatantsfrom digestions I and II, the calvariae were treated further two timeswith 3 ml of 2 mg/ml collagenase (30 mins, 37° C.). The supernatants ofdigestions III and IV were pooled, centrifuged, and the cells washed inDulbecco's modified Eagle's medium (DME) with 10% fetal calf serum(FCS), suspended in DME/10% FCS, and placed in 75 Cm³ flasks. The cellswere incubated under 5% CO₂ and 95% air at 37° C. Confluence was reachedby 5-6 days, at which time the cells were subcultured. Aftertrypsinization using trypsin-EDTA (0.05%/0.53 mM), the cells were rinsedin minimum essential medium (MEM) with 5% FCS and resuspended in a freshmedium, then seeded at 5×10⁴ cells/ml in 24-well plates (0.5 ml cellsuspension per well, i.e., 1.4×10⁴ cells/cm 2). The osteoblast-likecharacter of these cells has been established by demonstration of highlevels of alkaline phosphatase activity and osteocalcin production [asdescribed by Groot, et al. (1985) Cell Biol Int Res 9, 528] and asensitive adenylate cyclase response to parathyroid hormone andprostaglandins [as described by Hermann-Erlee, et al. (1986) NinthInternational Conference on calcium regulating hormones and bonemetabolism, p 409].

[0025] Proliferation studies (cell counts and thymidine incorporation)were performed both in actively growing and non-actively growing cellpopulations. To produce actively growing cells, sub-confluentpopulations (24 h after subculturing) were placed in fresh MEMcontaining 1% FCS and a lactoferrin sample. To produce non-activelygrowing cells, sub-confluent populations were placed in serum-freemedium with 0.1% bovine serum albumin plus a lactoferrin sample. Cellnumbers were analyzed at 6, 24, and 48 hours after the addition oflactoferrin samples (i.e., purified lactoferrin, holo-lactoferrin, andapo-lactoferrin) prepared as described above. The cell numbers weredetermined after detaching cells from the wells by exposure totrypsin/EDTA (0.05%/0.53 mM) for approximately 5 minutes at 37° C.Counting was performed in a haemocytometer chamber. [³H]-thymidineincorporation into actively growing and non-actively growing cells wasassessed by pulsing the cells with [³H]-thymidine (1 μCi/well) two hoursbefore the end of the incubation. The experiment was terminated at 6,24, or 48 hours by washing the cells in MEM containing cold thymidinefollowed by the addition of 10% trichloroacetic acid. The precipitatewas washed twice with ethanol:ether (3:1), and the wells desiccated atroom temperature. The residue was redissolved in 2 M KOH at 55° C. for30 min, neutralized with 1 M HCl, and an aliquot counted forradioactivity. For both cell counts and thymidine incorporation, eachexperiment at each time point was performed at least 4 different timesusing experimental groups consisting of at least 6 wells.

[0026] The mitogenic response of the purified lactoferrin sample wasfound to be very potent, as shown by a markedly increased rate ofosteoblast cell proliferation (i.e., increase in thymidine incorporationinto DNA of growing cells). The potent osteogenic response seen abovewas compared with that of insulin-like growth factor 1 (IGF-1), awell-recognized osteoblast mitogen. IGF-1 showed a maximal effect of1.25 times the control in the same osteoblast cell culture system,whereas lactoferrin's effect was 2.26 times that of the control for thehighest dose tested (10 μg/ml).

[0027] Lactoferrin Promotes Proliferation of Chondrocytes

[0028] Chondrocytes were isolated by removing cartilage (full-depthslices) from the tibial and femoral surfaces of sheep under asepticconditions. Slices were placed in Dulbecco's Modified Eagles (DME) mediacontaining 5% FBS (v/v) and antibiotics (penicillin 50 g/L, streptomycin50 g/L and neomycin 100 g/L) and chopped finely with a scalpel blade.Tissue was removed and incubated at 37° C. with firstly pronase (0.8%w/v for 90 minutes) followed by collagenase (0.1% w/v for 18 hours) tocomplete the digestion. Cells were isolated from the digest bycentrifugation (10 minutes at 1300 rpm), resuspended in DME/5% FBS,passed through a nylon mesh screen of 90 Fm pore size to remove anyundigested fragments, and recentrifuged. The cells were then washed andresuspended twice in the same media, seeded into a 75 cm² flaskcontaining DME/10% FBS, and incubated under 5% CO²/95% air at 37° C.Confluence was reached by 7 days, at which time the cells weresubcultured. After trypsinization using trypsin-EDTA (0.05%/0.53 mM),the cells were rinsed in DME/5% FBS and resuspended in a fresh medium,then seeded into 24-well plates (5×10⁴ cells/mL, 0.5 mL/well).Measurement of thymidine incorporation was performed in growth-arrestedcell populations as for the osteoblast-like cell cultures describedabove. Lactoferrin was found to stimulate chondrocyte proliferation atconcentrations above 0.1 μg/ml.

[0029] Lactoferrin Promotes Proliferation of Osteoblasts in OrganCulture

[0030] Neonatal mouse organ culture has been previously described(Cornish, et al. (1998) Am J Physiol 274, E827-E833). Briefly, two-dayold neonatal mice were subcutaneously injected with radioactivelylabeled ⁴⁵Ca. Three days later, the calvariae were excised and placed onmesh grids in Petri dishes containing 0.1% bovine serum albumin/Media199. Lactoferrin was added, and the calvariae were incubated for 48hours. Four hours before the end of the incubation period,[³H]-thymidine was added. The experiment was terminated, and ⁴⁵Carelease and thymidine incorporation were measured. Lactoferrin was foundto stimulate DNA synthesis, which reflects the proliferation of cells ofthe osteoblast lineage.

[0031] Lactoferrin Signals Via MAP Kinase in Osteoblasts

[0032] This methodology has been previously described (Grey, et al.(2001) Endocrinology 142, 1098-1106). Specifically, primary ratosteoblasts prepared as described above were seeded in 6-well tissueculture plates at an initial density of 5×10⁴ cells/ml in MEM 5% FCS,and grown to 80-90% confluence. After serum starvation overnight, cellswere treated at room temperature with lactoferrin in MEM/0.1% BSA. Inexperiments designed to determine the effect of inhibitors of signaltransduction on lactoferrin-induced p42/44 MAP kinase phosphorylation,the cells were pre-treated with the inhibitor for 30 min prior toaddition of lactoferrin. After treatment for the indicated period oftime, the treatment medium was aspirated, the cells were washed inice-cold PBS and then scraped in ice-cold HNTG lysis buffer (50 mMHEPES, pH 7.5, 150 mM NaCl, 1% Triton, 10% glycerol, 1.5 mM MgCl₂, 1 mMEDTA) containing a cocktail of protease and phosphatase inhibitors (1 mMPMSF, 1 μg/ml peptatin, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mMsodium vanadate, 500 mM NaF). The lysates were briefly vortexed,centrifuged at 13,000 rpm at 4° C., then stored at −70° C. untilanalyzed. Protein content of the cell lysates was measured using the DCprotein assay (BioRad, Hercules, Calif.). Equal amounts of the wholecell lysate (30-50 μg) were subjected to 8% SDS-PAGE, transferred tonitrocellulose membranes, and immunoblotted overnight at 4° C. with ananti-phospho-p42/44 MAP kinase antibody (1:1000). As a control forprotein loading, the same filters were stripped and re-probed with anantibody against total p42/44 MAP kinase (1:400). Incubation with theHRP-conjugated secondary antibody was for 1 h at room temperature, andthe membranes were analyzed by ECL. Immunoblots were repeated at least 3times. Lactoferrin was found to induce phosphorylation of p42/p44 MAPkinases in osteoblasts in a dose- and time-dependent manner atconcentrations of 1-100 μg/ml.

[0033] Lactoferrin Stimulates Bone Growth in Vivo

[0034] The mouse model used in these studies have been previouslydescribed (Cornish, et al. (1993) Endocrinology 132, 1359-1366).Injections (0 mg, 0.04 mg, 0.4 mg and 4 mg) of lactoferrin were givendaily for 5 days, and the animals were sacrificed one week later. Boneformation was determined by fluorescent labeling of newly formed bone.Indices of bone resorption and of bone mass were determined byconventional light microscopy, assisted by image analysis software.Local injection of lactoferrin in adult mice resulted in increasedcalvarial bone growth, with significant increases in bone area afteronly 5 injections. and the animals were sacrificed one week later. Boneformation was determined by fluorescent labeling of newly formed bone.

[0035] Application 1

[0036] Set yoghurts of between 14 and 17% solids, with or without fruitadded, can be prepared as follows:

[0037] Medium heat skim milk powder (between 109-152 g) and ALACOstabilizer (100 g) are reconstituted with approximately 880 ml of 50° C.water. Anhydrous Milk Fat (20 g) is then added and mixed for 30 min. Themixture is then heated to 60° C., homogenized at 200 bar, and thenpasteurized at 90° C. After cooling to a temperature between 40-42° C.,a starter mixture and the freeze-dried protein preparation describedabove (up to 50 mg of lactoferrin at 95% purity or an equivalentquantity from a not so highly purified source) is added. If desired,fresh fruit may also be added at this point. The mixture is then filledinto containers, incubated at 40° C. until pH 4.2-4.4 is reached, andthen chilled in a blast cooler.

[0038] An alternative method for preparing the same set yoghurts is bydry blending the indicated quantity of lactoferrin or the indicatedquantity as a dose rate, into the dry milk solids, prior to its use inthe yoghurt formulation.

[0039] Application 2

[0040] Dry blends of either skim or whole milk powder with calcium andthe freeze dried lactoferrin preparations can give dairy basedformulations or compositions which can be used either as functionalfoods or as functional food ingredients. Such compositions can be usedas reconstituted milks, milk powder ingredients, dairy desserts,functional foods, cheeses or butter or beverages, and nutraceuticals ordietary supplements. Blending the dry ingredients in ratios of milkpowder:calcium:active lactoferrin agent between 90:9.5:0.5 and94:5.95:0.0001 provide compositions suitable for such uses.

[0041] Application 3

[0042] Blended compositions of milk powder, calcium, and the lactoferrinrich ingredient can be used as bone health functional foods, bone healthfood ingredients, or as a food ingredient for delivery of bone healthnutrients in a range of health foods.

[0043] For such compositions, the calcium and protein contents of thecompositions need to be adjusted to required, allowable nutritionallimits. Commercially available ingredient milk powders typicallycontains between 300 and 900 mg calcium per 100 g powder, depending upontheir sources. A source of calcium may be added to the powder to extendthe calcium content up to 3% by weight of the ingredient milk powder asa blend. The protein level of commercially available ingredient milk ordairy-based protein powders varies depending upon the type of theingredient, the method of its manufacture, and its intended use.Ingredient milk powder typically contains between 12% and 92% protein.Examples are commercially available skim and whole milk powders, foodgrade caseins, caseinates, milk protein concentrate powders, spray driedultrafiltered or microfiltered retentate powders, and the milk proteinisolate products. The lactoferrin rich preparation may be incorporatedinto a protein and calcium blend to give nutritional milk powders thatcan be used as ingredients in healthy foods and drinks. Such blendsprovide ingredients suitable for use in preparing yoghurts and yoghurtdrinks, acid beverages, ingredient milk powder blends, pasteurizedliquid milk products, UHT milk products, cultured milk products,acidified milk drinks, milk-and-cereal combination products, maltedmilks, milk-and-soy combination products. For such uses, the blend canhave a composition where the calcium content is between 0.001% and 3.5%(w/w), the protein composition is between 2% and 92%, and lactoferrin asthe osteoblast proliferating agent is added at levels between 0.000001%and 5.5%.

Other Embodiments

[0044] All of the features disclosed in this specification may becombined in any combination. Each feature disclosed in thisspecification may be replaced by an alternative feature serving thesame, equivalent, or similar purpose. Thus, unless expressly statedotherwise, each feature disclosed is only an example of a generic seriesof equivalent or similar features.

[0045] From the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, other embodiments are also within the scope of thefollowing claims.

What is claimed is:
 1. A pure lactoferrin polypeptide containing no morethan two metal ions per molecule, wherein the polypeptide stimulatesskeletal growth and inhibits bone resorption.
 2. The lactoferrinpolypeptide of claim 1, wherein the polypeptide contains two metal ionsper molecule.
 3. The lactoferrin polypeptide of claim 2, wherein thepolypeptide contains iron ion, copper ion, chromium ion, cobalt ion,manganese ion, zinc ion, or magnesium ion.
 4. The lactoferrinpolypeptide of claim 3, wherein the polypeptide contains iron ion. 5.The lactoferrin polypeptide of claim 1, wherein the polypeptide containsiron ion, copper ion, chromium ion, cobalt ion, manganese ion, zinc ion,or magnesium ion.
 6. The lactoferrin polypeptide of claim 5, wherein thepolypeptide contains iron ion.
 7. A mixture comprising a lactoferrinpolypeptide of claim 1 and a fragment thereof.
 8. The mixture of claim7, wherein the polypeptide contains two metal ions per molecule.
 9. Themixture of claim 8, wherein the polypeptide contains iron ion, copperion, chromium ion, cobalt ion, manganese ion, zinc ion, or magnesiumion.
 10. The mixture of claim 9, wherein the polypeptide contains ironion.
 11. The mixture of claim 7, wherein the polypeptide contains ironion, copper ion, chromium ion, cobalt ion, manganese ion, zinc ion, ormagnesium ion.
 12. The mixture of claim 11, wherein the polypeptidecontains iron ion.
 13. The mixture of claim 7, wherein the mixturecomprises a hydrolysate of a lactoferrin polypeptide of claim
 1. 14. Anutraceutical composition comprising a lactoferrin polypeptide ofclaim
 1. 15. The nutraccutical composition of claim 14, wherein thepolypeptide contains two metal ions per molecule.
 16. The nutraceuticalcomposition of claim 14, wherein the polypeptide contains iron ion,copper ion, chromium ion, cobalt ion, manganese ion, zinc ion, ormagnesium ion.
 17. The nutraceutical composition of claim 16, whereinthe polypeptide contains iron ion.
 18. A neutraceutical compositioncomprising a mixture of claim
 7. 19. The nutraceutical composition ofclaim 18, wherein the polypeptide contains two metal ions per molecule.20. The nutraceutical composition of claim 19, wherein the polypeptidecontains iron ion, copper ion, chromium ion, cobalt ion, manganese ion,zinc ion, or magnesium ion.
 21. The nutraceutical composition of claim20, wherein the polypeptide contains iron ion.
 22. A pharmaceuticalcomposition comprising a lactoferrin polypeptide of claim 1 and apharmaceutically acceptable carrier.
 23. The pharmaceutical compositionof claim 22, wherein the polypeptide contains two metal ions permolecule.
 24. The pharmaceutical composition of claim 23, wherein thepolypeptide contains iron ion, copper ion, chromium ion, cobalt ion,manganese ion, zinc ion, or magnesium ion.
 25. The pharmaceuticalcomposition of claim 24, wherein the polypeptide contains iron ion. 26.A pharmaceutical composition comprising a mixture of claim 7 and apharmaceutically acceptable carrier.
 27. The pharmaceutical compositionof claim 26, wherein the polypeptide contains two metal ions permolecule.
 28. The pharmaceutical composition of claim 26, wherein thepolypeptide contains iron ion, copper ion, chromium ion, cobalt ion,manganese ion, zinc ion, or magnesium ion.
 29. The pharmaceuticalcomposition of claim 28, wherein the polypeptide contains iron ion. 30.A method of treating a bone-related disorder, the method comprisingadministering to a subject in need thereof an effective amount of alactoferrin polypeptide of claim
 1. 31. The method of claim 30, whereinthe polypeptide contains two metal ions per molecule.
 32. The method ofclaim 30, wherein the polypeptide contains iron ion, copper ion,chromium ion, cobalt ion, manganese ion, zinc ion, or magnesium ion. 33.The method of claim 32, wherein the polypeptide contains iron ion.
 34. Amethod of treating a bone-related disorder, the method comprisingadministering to a subject in need thereof an effective amount of amixture of claim
 7. 35. The method of claim 34, wherein the polypeptidecontains two metal ions per molecule.
 36. The method of claim 34,wherein the polypeptide contains iron ion, copper ion, chromium ion,cobalt ion, manganese ion, zinc ion, or magnesium ion.
 37. The method ofclaim 36, wherein the polypeptide contains iron ion.